Niabella beijingensis sp. nov. and Thermomonas beijingensis sp. nov., two bacteria from constructed wetland

Two Gram-stain-negative, aerobic, non-motile and rod-shaped bacterial strains designated 3A5MI-3T and RSS-23T were isolated from the Dragon-shaped Wetland System in Beijing Olympic Park, PR China. Strain 3A5MI-3T grew at 15–45 °C, pH 5.0–9.0 and with 0–2 % NaCl (w/v), and strain RSS-23T grew at 15-40 oC, pH 5.5–9.0 and with 0–1 % NaCl (w/v). Phylogenetic analyses of 16S rRNA gene sequences revealed that strains 3A5MI-3T and RSS-23T were members of Bacteroidetes and Proteobacteria , respectively. Phylogenetically closest relatives of strains 3A5MI-3T and RSS-23T were Niabella pedocola R384T and Thermomonas aquatica SY21T, respectively. The cells of strain 3A5MI-3T contained menaquinone MK-7 and phosphatidylethanolamine, and the major cellular fatty acids were composed of iso-C15 : 0, iso-C15 : 1 ω6c and/or iso-C15 : 1 ω7c, iso-C17 : 0 3-OH, C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Strain RSS-23T contained ubiquinone Q-8 and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unknown phospholipids and an unknown lipid, and its major cellular fatty acids were iso-C15 : 0, iso-C17 : 1 ω9c, iso-C11 : 0 3-OH and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). DNA sequencing resulted in 6.59 Mb for the strain 3A5MI-3T genome and 2.79 Mb for the strain RSS-23T genome. The calculated G+C molar contents for strains 3A5MI-3T and RSS-23T were 47.07 and 61.21 mol%, respectively. According to phenotypic and phylogenetic characteristics, strains 3A5MI-3T and RSS-23T represent novel species of the genera Niabella and Thermomonas for which the names Niabella beijingensis sp. nov. and Thermomonas beijingensis sp. nov. are proposed. The type strain for N. beijingensis sp. nov. is 3A5MI-3T (=CGMCC 1.17737T=KCTC 82817T). The type strain for T. beijingensis sp. nov. is RSS-23T (=CGMCC 1.17738T=KCTC 82820T).

In this report, we describe two bacterial strains that were isolated from a constructed wetland system, by phylogenetic, physiological, biochemical, and genomic analyses. The constructed wetland system, also called the Dragon-shaped Wetland, located in the central area of the Beijing Olympic Park area (40° 0.2′ N and 116° 22.28′ E), is the largest urban artificial water system in Asia. The water in the Dragon-shaped Water System comes mainly from the Beijing Qinghe Water Reclamation Plant, and the entire water system has a complete circulation system. Sludge samples from the Dragon-shaped Wetland water system were collected, and were serially diluted with 0.85 % NaCl (w/v) and plated onto Reasoner's 2A (R2A) agar (Difco). The isolates, designated as 3A5MI-3 T and RSS-23 T , were obtained after incubation for 3 days at 30 °C and were routinely stored at −80 °C as suspensions in R2A broth supplemented with 20 % (v/v) glycerol.
Genomic DNA was extracted with a commercial TIANamp Bacteria DNA Kit, and 16S rRNA genes were amplified by PCR with universal bacterial primers 27 F and 1492R [20], which was also used for sequencing the PCR product. The almost-complete sequence was compared with 16S rRNA gene sequences from GenBank. The 16S rRNA gene sequences were aligned using ClustalW [21]. Phylogenetic analyses were carried out using three phylogenetic algorithms: neighbour-joining [22], maximumlikelihood [23] and maximum-parsimony [24]. Phylogenetic trees were reconstructed and bootstrapped with 1000 replicates of each sequence using mega version 7.0 [25]. The CVTree method was used to reconstruct phylogenomic tree based on whole genomes [26].
Analysis of 16S rRNA gene sequences in GenBank indicated that strain 3A5MI-3 T was phylogenetically close to Niabella pedocola R384 T (95.97 %), 'Niabella thaonhiensis' NHI-24 T  and Niabella ginsengisoli GR 10-1 T (92.45 %), as well as to Terrimonas rhizosphaerae CR94 T (92.14 %) and was <92 % similar to some other species included in this analysis ( Fig. 1a and S1, available in the online version of this article). Based on the results of phylogenetic analysis and 16S rRNA gene identity, N. pedocola JCM 31011 T was selected as a reference strain for phenotypic tests (see following paragraph).
To analyse the genomic properties of strains 3A5MI-3 T and RSS-23 T , whole-genome sequencing was performed using the Illumina system. The genomic assembly was performed with the SPAdes software (version 3.9.0) using clean data [27]. Average nucleotide identity (ANI) values were calculated with ChunLab's online ANI Calculator [28]. Digital DNA-DNA hybridization (dDDH) was performed by using the Genome-to-Genome Distance Calculator (3.0) [29].
The genome of strain 3A5MI-3 T contained three contigs, and the total length was 6.59 Mb, encoding 6358 genes. The calculated DNA G+C content of strain 3A5MI-3 T was 47.07 mol%. The genome of strain 3A5MI-3 T carries 49 ncRNA genes, including three rRNA operons (5S, 16S, 23S), 41 tRNA genes and five sRNA genes. In addition, there is a clustered regularly interspaced short palindromic repeat sequence (CRISPR) with a length of 2862 bp in this genome. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional category distribution revealed that large numbers of genes in the genome of strain 3A5MI-3 T belonged to the metabolism (61 %), genetic information processing (16 %), environmental information processing (7 %) and cellular processes (7 %). Annotated with the RefSeq non-redundant proteins (NR) database, the genomes of strain 3A5MI-3 T harboured nine genes encoding phosphohydrolase, nine genes encoding phosphatase, 11 genes encoding glucosidase, 51 genes encoding glycosyl hydrolase families, 36 genes encoding starch binding protein, six genes encoding polysaccharide lyase, seven genes encoding polysaccharide deacetylase, 28 genes encoding SusC/RagA family TonB-linked outer membrane protein, 13 genes encoding xylanase, one gene encoding cellulase and one gene encoding chondroitinase-B. We found genes for xylanase and cellulase in the strain 3A5MI-3 T and N. pedocola JCM31011 T genomes. APIZYM and carbon source utilization experiments confirmed that strain 3A5MI-3 T and N. pedocola JCM31011 T could utilize xylan and cellulose. Although strain 3A5MI-3 T and N. pedocola JCM31011 T both have the chitinase gene in their genome, neither of them can use chitin. Trypsin-like serine proteases were found in the strain 3A5MI-3 T genome but not in strain N. pedocola JCM31011 T , which corresponds to APIZYM trypsin activity for strain 3A5MI-3 T but not for N. pedocola JCM31011 T . Comparing the genomes of strain 3A5MI-3 T and N. pedocola JCM 31011 T , we found that the specific genes detected in strain 3A5MI-3 T were as follows: four genes encoding lipopolysaccharide biosynthesis protein, three genes encoding biotin synthase, three genes encoding CRISPR-associated protein Cas1, two genes encoding 2-dehydropantoate 2-reductase, one gene encoding bifunctional NAD(P)H-nitrite reductase/anaerobic dehydrogenase, two genes encoding arabinogalactan endo-1,4-beta-galactosidase, three genes encoding TDP-4-oxo-6-deoxy-d-glucose aminotransferase, one gene encoding zinc-binding alcohol dehydrogenase family protein, three genes encoding 3-phytase, three genes encoding alpha-glucuronidase and one gene encoding Alg9-like mannosyltransferase family protein. The accession numbers of the sequences in the database relating to the specific or characteristic (marked in yellow) genes of strain 3A5MI-3 T and N. pedocola JCM 31011 T are provided as supplementary material. The ANI value between strains 3A5MI-3 T and N. pedocola JCM 31011 T was calculated using ChunLab's online ANI Calculator [28]. We performed genome sequencing of N. pedocola JCM31011 T and its GenBank/ EMBL/DDBJ accession number is JAJNEC000000000. The ANI result was 77.48 %, below the 95 % inter-species threshold [30].   [7]. +, Positive; −, negative; w, weakly positive. The data of strain 3A5MI-3 T and N. pedocola JCM 31011 T were taken from the current study, others were taken from the published literature. The genome of strain RSS-23 T contained also three contigs, and the total length is 2.79 Mb, encoding 2575 genes. The calculated genomic DNA G+C content of strain RSS-23 T was 61.21 mol%. The genome of strain RSS-23 T carries 58 ncRNA genes, including six rRNA genes (5S, 16S, 23S), 49 tRNA genes and three sRNA genes. The KEGG functional category distribution revealed that large numbers of genes in the genome of strain RSS-23 T belonged to the metabolism (53 %), genetic information processing (16 %), environmental information processing (14 %), cellular processes (8 %). Annotated with the RefSeq NR database, the genomes of strain RSS-23 T harboured 61 genes encoding phosphatase, six genes encoding glucosidase, four genes encoding glycosyl hydrolase, 27 genes encoding glycosyl transferase and 11 genes encoding polysaccharide biosynthesis protein. APIZYM positive enzyme genes such as alkaline phosphatase, acid phosphatase and α-glucosidase were also found in the genome. Comparing the genomes of strains RSS-23 T and T. fusca DSM 15424 T , we found that the specific genes contained in strain RSS-23 T are as follows: three genes encoding aminotransferase class I/II, six genes encoding chloride channel protein, three genes encoding penicillin acylase, three genes encoding UDP-glucose 4-epimerase, three genes encoding UDP-N-acetylglucosamine 2-epimerase and three genes encoding carbonate dehydratase. The accession numbers of the sequences on the database relating to the specific or characteristic (marked in yellow) genes of strain RSS-23 T and T. fusca DSM 15424 T are provided as supplementary files. The ANI values between strain RSS-23 T and T. fusca DSM 15424 T or T. aquatica SY21 T were 78.10 and 76.11 %, respectively, which were below the 95 % inter-species threshold [30]. The estimated dDDH values of strain RSS-23 T to T. fusca DSM 15424 T and T. aquatica SY21 T were 25.20 and 20.90 %, respectively, which were below the standard value generally recommended for species differentiation.
The morphology and size of cells grown on R2A agar at 30 °C for 2 days were observed by using transmission electron microscopy (JEM-1400, jeol), and motility was observed with light microscopy. Gram-staining was performed according to Hucker [31].
The cellular fatty acids were determined using cells grown on R2A agar for 2 days at 30 °C with the standard midi protocol (Sherlock Microbial Identification System, version 6.0B) and analysed with a gas chromatograph (GC6890, Agilent) [33]. The extraction, purification and analysis of quinones were carried out according to the methods of Collins et al. [34]. The total lipids were extracted using chloroform and methanol, followed by thin-plate biphasic chromatography to identify each lipid component [35].
The composition of the fatty acids is shown in Table 3. The major cytoquinone was determined to be MK-7, as previously reported *iso-C 15 : 1 F should correspond to either iso-C 15 : 1 ω6c and/or iso-C 15 : 1 ω5c. The double bond position is presumptive [36]. †Summed features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The midi system groups these fatty acids together as one feature with a single percentage of the total. Summed feature 3 comprised C 16 : 1 ω7c/C 16 : 1 ω6c.
for described members of the genus Niabella. The polar lipid profile of strain 3A5MI-3 T contained phosphatidylethanolamine and three unknown lipids (Fig. S4a).
Strain RSS-23 T also contained aliphatic saturated fatty acid (iso-C 15 : 0 ) as its predominant cellular fatty acid, which accounted for about one third of total fatty acids. The fatty acid profiles are shown in Table 4. The major isoprenoid quinone was determined to be Q-8, as previously reported for members of the genus Thermomonas. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unknown phospholipids and an unknown lipid (Fig. S4b).
Based on the phylogenetic and phenotypic characteristics, we conclude that strains 3A5MI-3 T and RSS-23 T represent novel species of the genera Niabella and Thermomonas, respectively, for which the names Niabella beijingensis sp. nov. and Thermomonas beijingensis sp. nov. are proposed.
The type strain, RSS-23 T (=CGMCC 1.17738 T =KCTC 82820 T ), was isolated from soil of the Dragon-shaped Wetland System in Beijing Olympic Park, PR China. The DNA G+C content of the type strain is 61.21 mol%.

Funding information
This work was financially supported by National Natural Science Foundation of China (Grant No. 31861133002).